Targeted vectors for cancer immunotherapy

ABSTRACT

This invention provides compositions and methods for treating cancer. More specifically this invention is directed to a targeted retroviral vector comprising a cytokine gene that can be administered either alone or in combination with a targeted retroviral vector comprising a cytocidal gene for treating cancer in a subject. Also provided are a kit or drug delivery system comprising the compositions for use in the methods described.

1. RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) ofprovisional application Ser. No. 60/250,185 filed. Nov. 29, 2000, thedisclosure of which is hereby incorporated by reference in its entirety.

2. FIELD OF THE INVENTION

This invention is in the field of oncology, more specifically thisinvention relates to targeted injectable vectors, such as targetedretroviral particles, for use in cancer to immunotherapy.

3. BACKGROUND OF THE INVENTION

Immune modulation in conjunction with tumor antigen presentation is apromising approach for optimizing the efficacy of cancer gene therapyprotocols for metastatic cancer or minimal residual disease. In tumorvaccine strategies, cytokines such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) are employed to recruitantigen-presenting cells, including dendritic cells and macrophages,which result in the activation of cytotoxic T lymphocytes (CTL) againstproteins expressed by cancer cells (Warren and Weiner, 2000; Kim et al.,2000). GM-CSF induces activation, proliferation, and differentiation ofa variety of immunologically active cell populations, therebyfacilitating the development of both humoral and cellular-mediatedimmunity (Warren and Weiner, 2000). One promising vaccine approachinvolves the insertion of the GM-CSF gene into autologous cancer cellsthat are then used for immunization (Jaffee, 1999; Suh et al., 1999).These genetically engineered tumor cells produce the GM-CSF protein inthe local environment of tumor cells, thereby activating the patients Tcells, which then function to eradicate the cancer at metastatic sites.Whether delivered as genetically engineered tumor cells or as thesoluble GM-CSF protein, the cytokine must be present in the same site asthe vaccine component (Mellstedt et al., 1999). Indeed, theestablishment of specific and long lasting antitumor immunity followingvaccination with GM-CSF tumor cells requires the simultaneous presenceof GM-CSF and tumor antigens at the vaccination site (Nagai et el.,1998). However, in spite of the therapeutic potential demonstrated inanimal models and early-phase clinical trials, the clinical developmentof these protocols has been limited by difficulties relating to theestablishment of autologous tumor cell cultures (Fong et al., 2000) andthe performance of individualized gene transfer procedures ex vivo(Borrello et al., 1999). Alternatively, local delivery of GM-CSF bydirect intratumoral injection, as well as paracrine secretion bygenetically engineered cells, has been shown to be more effective inupregulating lymph node sensitization when compared to systemicadministration (Kurane et al., 1997).

A novel cancer immunotherapy approach for metastatic cancer wouldexploit the potential of systemically administered matrix-targetedretroviral vectors, infused sequentially intravenously, to efficientlydeliver both a cytokine gene and/or a cytocidal construct to tumor cellsand associated tumor vasculature. The efficacy of matrix-targeted genedelivery has been demonstrated in models of liver metastasis (Gordon etal., 2000a) and subcutaneous human cancer xenografts in nude mice(Gordon et al., 2000b). The present invention provides targetedretroviral paticles, for systemic administration, carrying one or morecytokine genes that provide high level efficiency of cytokine genedelivery into a tumor and recruitment of host mononuclear cells (tumorinfiltrating lymphocytes) into the tumor.

4. SUMMARY OF THE INVENTION

This invention relates in general to compositions and methods for use incancer immunotherapy. More particularly this invention is directed totargeted injectable vectors for use in cancer immunotherapy.

It is an object of this invention to provide a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector and a cytokine gene.

It is an object of this invention to provide a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector to an extracellular matrix component or tumor vasculature and acytokine gene.

It is a further object of this invention to provide a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle to an extracellular matrix componentcomprising a binding region which binds to an extracellular matrixcomponent and a cytokine gene (e.g., granulocyte-macrophagecolony-stimulating factor (GM-CSF)).

It yet another object of this invention to provide a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector particle (e.g., targeting an extracellular matrix component ortumor vasculature) and a cytokine gene (e.g., GM-CSF) and a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle (e.g., targeting an extracellular matrixcomponent or tumor vasculature) and a cytocidal gene (e.g., tumorsuppressor genes).

It is another object of this invention to provide a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector (e.g., targeting an extracellular matrix component or tumorvasculature) and a cytokine gene (e.g., GM-CSF) and/or a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle (e.g., targeting an extracellular matrixcomponent or tumor vasculature) and a cytocidal gene (e.g., tumorsuppressor genes) to be administered intravenously or intra-arterially.

Yet another object of this invention is a method of treating cancer in asubject comprising administering a targeted retroviral particlecomprising a modified viral surface protein for targeting the vector anda cytokine gene either alone or in conjunction with a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle and a cytocidal gene.

It is a further object of this invention to provide a method of treatingprimary tumors or tumors located in surgically inaccessible sites in asubject comprising administering a targeted retroviral particlecomprising a modified viral surface protein for targeting the vector anda cytokine gene either alone or in conjunction with a targetedretroviral particle comprising a modified viral surface protein fortargeting the vector particle and a cytocidal gene.

It is yet another object of this invention to provide methods ofadministering targeted injectable vectors, such as targeted retroviralparticles to achieve high level efficiency of cytokine gene deliveryinto distant tumor sites resulting in secretion of GM-CSF by transducedtumor cells within the solid tumor and/or recruitment of host immunecells (e.g., mononuclear cells), such as tumor infiltrating lymphocytes(TIL) into the tumor.

It is a further object of this invention to provide a kit or drugdelivery system comprising the compositions for use in the methodsdescribed herein.

5. BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a schematic diagram of the molecular engineering of theGM-CSF retroviral expression vector. The retroviral expression vector(pREX II) was created by engineering a multiple cloning site (MSC) intothe G1XSvNa vector (Genetic Therapy, Inc.) to produce G1 (MCS)SvNa (A),which is then subjected to Kpn I digestion followed by fusion of the KpnI fragment (C) with the linearized pRV109 vector (B). The resulting pREXII retroviral expression vector (D) is driven by a hybrid CMV/MSV/MLVpromoter at the 5′ LTR and a standard MLV LTR at the 3′ end. The 0.44 kbcDNA encoding human granulocyte macrophage colony stimulating factor(GM-CSF), was cloned into the unique Not 1 (5,) and Xho 1 (3,) cloningsites of the pREX II vector (E).

FIG. 2 shows the immunologic detection of human GM-CSF protein secretedby transduced NIH3T3 cells and transfected 293T cells. (A) The darkenedELISA wells indicate immunoreactive human GM-CSF secreted by transducedNIH3T3 and transfected 293T cells, as detected by a polyclonal goatanti-human GM-CSF IgG (R&D Systems, Inc.). (B) Serial dilutions ofsupernatant collected from transduced NIH3T3 and transfected 293T cellcultures were used to measure immunoreactive human GM-CSF protein (shownas decreasing immunoreactivity with each dilution), against a purifiedrhGM-CSF standard.

FIG. 3 shows high level transduction of the tumor nodule by intravenousinjection of the matrix-targeted retroviral vector bearing a nucleartargeted β-galactosidase gene. (A: X 400) A negative staining controltumor nodule from Mx-null vector-treated animal. (B: X400) A tumornodule from a Mx-nBg vector-treated animal. β-galactosidase expressingtumor cells (closed arrows) and tumor endothelial cells (open arrows)are shown as reddish-brown nuclear stained cells, counterstained withmethyl green.

FIG. 4 is a histologic section of a tumor nodule expressingimmunoreactive human GM-CSF from nude mice treated with intravenousinjections of the matrix-targeted GM-CSF retroviral vector. (A: X 40) Anegative staining control tumor nodule from Mx-null vector-treatedmouse. (B: X40) A tumor nodule from a Mx-Gm-CSF vector-treated animal.Human GM-CSF expressing tumor cells are shown as reddish-brown nuclearstained cells, counterstained with methyl green. (C: X200) Boxed area inB with human GM-CSF expressing tumor cells are indicated by arrows. (D:X40) Tumor nodule in B as negative control with no primary antibody.

FIG. 5 shows extensive infiltration of the tumor nodule by hostmononuclear cells (tumor infiltrating lymphocytes) in mice treated withintravenous injections of the matrix-targeted GM-CSF retroviral vector.(A: 40X) H&E stained tissue section of tumor nodule from a Mx-nullvector-treated mouse. (B: X40) H&E stained tissue section of tumornodule from a Mx-GM-CSF vector-treated animal. (C: X100) Highermagnification of A (boxed area) showing a pleomorphic population oftumor cells, tumor stromal and endothelial cells with minimalmononuclear cell infiltration. (D: X100) Higher magnification of Bshowing extensive infiltration of host mononuclear cells.

6. DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA. These methods aredescribed in the following publications. See, e.g., Sambrook, et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) edition (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); “PCR: A PRACTICALAPPROACH” (M. MacPherson, et al., IRL Press at Oxford University Press(1991)); PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames andG. R. Taylor eds. (1995)); ANTIBODIES, A LABORATORY MANUAL (Harlow andLane, eds. (1988)); and ANIMAL CELL CULTURE (R. I. Freshney, ed.(1987)).

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a rtargeted retroviral particle”includes a plurality of particles, including mixtures thereof.

The term “cancer” includes a myriad of diseases generally characterizedby inappropriate cellular proliferation, abnormal or excessive cellularproliferation. Examples of cancer include but are not limited to, breastcancer, colon cancer, prostate cancer, pancreatic cancer, melanoma, lungcancer, ovarian cancer, kidney cancer, brain cancer, or sarcomas. Suchcancers may be caused by, chromosomal abnormalities, degenerative growthand developmental disorders, mitogenic agents, ultraviolet radiation(UV), viral infections, inappropriate tissue expression of a gene,alterations in expression of a gene, or carcinogenic agents.

The term “treatment” includes, but is not limited to, inhibition orreduction of proliferation of cancer cells, destruction of cancer cells,prevention of proliferation of cancer cells or prevention of initiationof malignant cells or arrest or reversal of the progression oftransformed premalignant cells to malignant disease or ameleriation ofthe disease.

The term “subject” refers to any animal, preferably a mammal such as ahuman. vetinary uses are also intended to be encompassed by thisinvention.

The present invention provides, in general, compositions and methods foruse in cancer immunotherapy. More particularly, this invention isdirected to targeted injectable vectors, such as targeted retroviralparticles, for use in cancer immunotherapy wherein the targetedretroviral particle comprises a modified viral surface protein fortargeting the vector particle and is capable of expressing a cytokinegene. These compositions and methods are based on an observation by theinventors that a targeted retroviral particles expressing a cytokinegene can be administered systemically and achieve (i) high levelefficiency of cytokine gene delivery into solid tumors, (ii) secretionof GM-CSF by tumor cells within the solid tumor, and (iii) recruitmentof host mononuclear cells (tumor infiltrating lymphocytes, TIL) into theGM-CSF secreting tumor nodules for eradication of primary, metastatic,inaccessible or minimal residual disease.

In one embodiment the invention provides a targeted injectable vector inthe form of a targeted retroviral particle comprising a cytokine gene.The retroviral particle is targeted to cancer cells or tumors bymodifying the viral surface protein to express a targeting polypeptide.Preferably, the targeting polypeptide recognizes motifs associated withcancer or tumor growth. Examples of motifs for recognition by thetargeting polypeptide include, but are not limited to, extra cellularmatrix (ECM) or tumor vasculature (see e.g., WO/01/07059 andWO/01/31036, hereby incorporated by reference). By way of example, thetargeting polypeptide could recognize the extra cellular matrix (ECM),endothelial cells or stromal cells exposed during tumor growth,angiogenesis or metastasis. Examples of collagen binding motifs that maybe used to target the ECM include, but are not limited to, the collagenbinding motif derived from the von Willebrand coagulation factor(Takigi, J., et al (1992) Biochemistry 32:8530 and WO/01/07059).

The targeted retroviral particle can be generated by conventionalmethods. By way of example, a three plasmid co-transfection system maybe used to construct the retroviral particle. Generally, in such asystem, one plasmid comprises the gag-pol genes, a second plasmidcomprises the chimeric or hybrid surface proteins for targeting theretroviral particle, and the third plasmid comprises an expressionvector containing the cytokine gene (e.g., Examples 1 and 2; Miller(1990) Human Gene Therapy Vol. 1, U.S. Pat. No. 5,952,225; Soneoka et al(1995) Nucl. Acid Research 23:628; WO/01/07059 and WO/01/31036, herebyincorporated by reference).

Expression vectors suitable for use in expressing the cytokine gene maycomprise at least one expression control element operably linked to thenucleic acid sequence encoding the cytokine gene. Expression controlelements may be inserted in the vector to control and regulate theexpression of the cytokine nucleic acid sequence. Examples of expressioncontrol elements include, but are not limited to, lac system, operatorand promoter regions of phase λ, yeast promoters, and promoters derivedfrom polyoma, adenovirus, retroviruses, or SV40. The vector may furthercomprise additional operational elements including, but not limited to,leader sequences, termination codons, polyadenylation signals, and anyother sequences necessary or preferred for the appropriate transcriptionand/or translation of the cytokine nucleic acid sequence. It will beunderstood by one skilled in the art that the correct combination ofrequired or preferred expression control elements will depend on thegene to be expressed, target tissue and the like. It will be furtherunderstood by one skilled in the art that such vectors are constructedusing conventional methodology (See e.g. Sambrook et al., (eds.) (1989)“Molecular Cloning, A laboratory Manual” Cold Spring Harbor Press,Plainview, N.Y.; Ausubel et al., (eds.) (1987) “Current Protocols inMolecular Biology” John Wiley and Sons, New York, N.Y.; WO/01/31036,hereby incorporated by reference) or are commercially available.

In a preferred embodiment, the targeted retroviral particles are viralvectors. Examples of viral expression vectors that may be used include,but are not limited to, retroviral vectors, such as lentivirus, vacciniavirus vectors, adenovirus vectors, herpes virus vector, or fowl poxvirus vector. In a preferred embodiment, the cytokine gene isincorporated into a retroviral expression vector and packaged into theretroviral particle (see e.g., WO/01/07059 and WO/01/31036, herebyincorporated by reference).

Cytokines modulate the immune system. The cytokine gene incorporatedinto the expression vector may be any cytokine gene, preferably acytokine which enhances or stimulates the humoral or cellular immuneresponse. The polynucleotide encoding the gene may be DNA or RNA. Thetargeted retroviral vector may comprise a polynucleotide encoding theentire cytokine protein or a polynucleotide encoding a portion of theprotein necessary for the biological activity of the cytokine. Cytokinesinclude but are not limited to, interleukins, lymphokines, monokines,interferons, colony stimulating factors and chemokines. Examples ofspecific cytokine that may be used include, but are not limited to,IL-1, TNF, IL-2, IFN-γ, IL-4, IL-7 and GM-CSF. Preferred cytokines areGM-CSF and IL-2. The targeted retroviral vectors may comprise all orpart of one or more cytokine genes.

In another embodiment this invention also provides a targeted retroviralparticle comprising a modified viral surface protein for targeting thevector (e.g., targeting an extracellular matrix component or tumorvasculature) and a cytocidal gene (e.g., tumor suppressor genes). Thesetargeted retroviral particles may be generated as described hereinabove, (see, e.g., WO/01/64870) The cytocidal gene may be any gene whichinhibits, destroys or prevents cancer cell growth or induces apoptosisin cancer cells. Examples of cytocidal genes includes, but is notlimited to, tumor suppressor genes (e.g., p53, RB), thymidine kinases(e.g., HSV thymidine kinase, CMV thymidine kinase) or mutated cyclin G1genes. The vector may comprise a polynucleotide encoding for an entirecytocidal gene or a portion of a polynucleotide encoding a portion ofthe cytocidal protein sufficient to exert its biological activity. Byway of example, the cytocidal gene may be a dominant negative mutationof the cyclin G1 protein (e.g., WO/01/64870). The targeted retroviralparticle comprising a modified viral surface protein for targeting thevector and a cytocidal gene may be used alone or in conjunction with thetargeted retroviral particle comprising a modified viral surface proteinfor targeting the vector and a cytokine gene. Alternatively, both thecytokine and cytocidal gene may be contained within the same targetedretroviral vector.

In an alternative embodiment, the targeted injectable vector may be inthe form of nonviral vectors, such as cationic liposomes expressing acytokine gene or cytocidal gene.

Another embodiment of this invention relates to methods of treatingcancer with immunotherapy by administering the targeted retroviralparticles described herein to a subject. The term treatment as usedherein is intended to include, but is not limited to, administration ofthe targeted retroviral particles prior to any evidence of disease(e.g., subjects at risk of occurrence of the disease or at risk ofrecurrence) or to mediate regression of the disease in a subject. Thequantity of targeted viral particle comprising a cytokine gene to beadministered is based on the titer of virus particles. By way ofexample, a range of particles to be administered is about 10⁹ to about10¹² colony forming units (cfu). After administration, the efficacy ofthe treatment can be assessed by cytokine production by transducedtumors, recruitment into the tumor site of immune cells (e.g., TIL) orantibodies that recognize the tumor antigen, and/or by tumor regression.One skilled in the art would know the conventional methods to assess theaforementioned parameters.

The targeted retroviral particle comprising the cytokine gene may beadministered alone or in conjunction with other therapeutic treatmentsor active agents. For example, the targeted retroviral particlecomprising a cytokine gene may be administered with the targetedretroviral particle comprising a cytocidal gene. The quantity of thetargeted retroviral particle comprising a cytocidal gene to beadministered is based on the titer of the virus particles as describedherein above. By way of example, if the targeted retroviral particlecomprising a cytokine gene is administered in conjunction with atargeted retroviral particle comprising a cytocidal gene the titer ofthe retroviral particle for each vector may be lower than if each vectoris used alone. The targeted retroviral particle comprising the cytokinegene may be administered concurrently or separately from the targetedretroviral particle comprising the cytocidal gene.

The methods of the subject invention also relate to methods of treatingcancer by administering a targeted retroviral particle (e.g., thetargeted retroviral vector expressing a cytokine either alone or inconjunction with the targeted retroviral vector expressing a cytocidalgene) with one or more other active agents. Examples of other activeagents that may be used include, but are not limited to,chemotherapeutic agents, anti-inflammatory agents, protease inhibitors,such as REV protease inhibitors, nucleoside analogs, such as AZT. Theone or more active agents may be administered concurrently or separately(e.g., before administration of the targeted retroviral particle orafter administration of the targeted retroviral particle) with the oneor more active agents. One of skill in the art will appreciate that thetargeted retroviral particle may be administered either by the sameroute as the one or more agents (e.g., the targeted retroviral vectorand the agent are both administered intravenously) or by differentroutes (e.g., the targeted retroviral vector is administeredintravenously and the one or more agents are administered orally).

An effective amount or therapeutically effective of the targetedretroviral particles to be administered to a subject in need oftreatment may be determined in a variety of ways. By way of example, theamount may be based on viral titer or efficacy in an animal model.Alternatively the dosing regimes used in clinical trials may be used asgeneral guidelines. The daily dose may be administered in a single doseor in portions at various hours of the day. Initially, a higher dosagemay be required and may be reduced over time when the optimal initialresponse is obtained. By way of example, treatment may be continuous fordays, weeks, or years, or may be at intervals with intervening restperiods. The dosage may be modified in accordance with other treatmentsthe individual may be receiving. However, the method of treatment is inno way limited to a particular concentration or range of the targetedretroviral particle and may be varied for each individual being treatedand for each derivative used.

One of skill in the art will appreciate that individualization of dosagemay be required to achieve the maximum effect for a given individual. Itis further understood by one skilled in the art that the dosageadministered to a individual being treated may vary depending on theindividuals age, severity or stage of the disease and response to thecourse of treatment. One skilled in the art will know the clinicalparameters to evaluate to determine proper dosage for the individualbeing treated by the methods described herein. Clinical parameters thatmay be assessed for determining dosage include, but are not limited to,tumor size, alteration in the level of tumor markers used in clinicaltesting for particular malignancies. Based on such parameters thetreating physician will determine the therapeutically effective amountto be used for a given individual. Such therapies may be administered asoften as necessary and for the period of time judged necessary by thetreating physician.

While it is possible for the targeted retroviral particle to beadministered in a pure or substantially pure form, it is preferable topresent it as a pharmaceutical composition, formulation or preparation.

Pharmaceutical compositions comprising the targeted retroviral particlesto be used in the methods described herein may be formulated andadministered by methods well known in the art (Remington'sPharmaceutical Sciences, 20th Edition, Lippincott, William & WilkinsBaltimore, Md.). For example, the compositions of the present inventionmay comprise an effective amount of the targeted retroviral particlesand a pharmaceutically and/or physiologically acceptable carrier. Thecharacteristics of the carrier will depend on the route ofadministration. Such a composition may contain, in addition to thetargeted retroviral particles, diluents, filters, salts, buffers,stabilizers, solubilizers and other materials well known in the art.

The compositions of the may be formulated for various routes ofadministration by methods well known in the art, including, but notlimited to parenteral administration, (e.g., for injection via theintravenous, intramuscular, sub-cutaneous, intratumoral orintraperitoneal routes), oral administration or topical administration.In a preferred embodiment, the targeted retroviral particles areadministered intravenously or intraarterially. Upon formulation, thecompositions are administered in a manner compatible with the dosageformulation. By way of example, the administration may vary from severaltimes a day to less frequent to administrations, such as once a day orevery other day for only a few days such as with a rest period ofvarying lengths, such as a week. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Veterinaryuses are also intended to be encompassed by this invention.

It is a further object of this invention to provide a kit or drugdelivery system comprising the compositions for use in the methodsdescribed herein. All the essential materials and reagents required foradministration of the targeted retroviral particle may be assembled in akit (e.g., packaging cell construct or cell line, cytokine expressionvector). The components of the kit may be provided in a variety offormulations as described above. The one or more targeted retroviralparticle may be formulated with one or more agents (e.g., achemotherapeutic agent) into a single pharmaceutically acceptablecomposition or separate pharmaceutically acceptable compositions.

The components of these kits or drug delivery systems may also beprovided in dried or lyophilized forms. When reagents or components areprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent, which may also be provided in another containermeans. The kits of the invention may also comprise instructionsregarding the dosage and or administration information for the targetedretroviral particle. The kits or drug delivery systems of the presentinvention also will typically include a means for containing the vialsin close confinement for commercial sale such as, e.g., injection orblow-molded plastic containers into which the desired vials areretained. Irrespective of the number or type of containers, the kits mayalso comprise, or be packaged with, an instrument for assisting with theinjection/administration or placement of the ultimate complexcomposition within the body of a subject. Such an instrument may be anapplicator, inhalant, syringe, pipette, forceps, measured spoon, eyedropper or any such medically approved delivery vehicle.

The following examples illustrate various aspects of the invention, butin no way are intended to limit the scope thereof.

7. EXAMPLES Example 1 Construction of the GM-CSF Retroviral ExpressionVector

The retroviral expression vector (pREX 11) was created by engineering amultiple cloning site (MSC) into the G1 XSvNa vector (Genetic Therapy,Inc.) to produce G1 (MCS)SvNa (FIG. 1A), which is then subjected to KpnI digestion followed by fusion of the Kpn I fragment (FIG. 1C) with thelinearized pRV109 vector (FIG. 1B). The resulting pREX II retroviralexpression vector (FIG. 1D) is driven by a hybrid CMV/MSV/MLV promoterat the 5′ LTR and a standard MLV LTR at the 3′ end. Bearing the strongCMV promoter and an SV40 ori, this plasmid is suitable for high titervector production in 293T cells prepared by transient transfectionprotocols. (Soneoka et al., 1995). The 0.44 kb cDNA encoding humangranulocyte macrophage colony stimulating factor (GM-CSF), GenBankaccession number NM 000758, flanked by PCR-derived restriction sites wascloned into the unique Not 1 (5′) and Xho 1 (3′) cloning sites of thepREX II vector (E).

Example 2 Production of Matrix-Targeted Retroviral Vectors Bearing aHuman GM-CSF Construct

High titer vectors were generated utilizing a transient three plasmidco-transfection system (Soneoka et al., 1995) in which the packagingcomponents gag-pol, a chimeric MLV-based env bearing a von Willebrandfactor-derived collagen-binding (matrix-targeting) motif expressed fromthe CMV promoter, and a retroviral vector bearing were placed onseparate plasmids, each containing the SV40 origin of replication. Theresulting vectors are referred to as Mx-GM-CSF, CAE-GM-CSF, Mx null, andMx-nBg to indicate the envelope and gene encoded in each vector.Mx-GM-CSF is a matrix (i.e. collagen)-targeted vector bearing a humanGM-CSF construct. CAE-GM-CSF, is a non-targeted vector bearing the wildtype MLV 4070A env protein (Morgan et al., 1993). Mx-null is amatrix-targeted vector bearing only the neo gene, and Mx-nBg, is acollagen-matrix-targeted vector bearing a nuclear targetedβ-galactosidase gene. The collagen-matrix-targeting results from theinsertion of a collagen-binding peptide derived from human vonWillebrand factor into the MLV 4070A env protein (Hall et al., 2000).Viral titers in murine NIH3T3 cells were determined as previouslydescribed, based on expression of the β galactosidase or neomycinphosphotransferase resistance, neo gene (Skotzko et al., 1995). Viraltiter was expressed as number of nuclear β-galactosidase expressingcolonies or G418 resistant colony forming units (cfu)/ml, and rangedfrom 0.3-1.8×10⁷ cfu/ml.

Example 3 GM-CSF Production in Transduced NIH3T3 and 293T Cell Cultures

To assess the production and secretion of GM-CSF, immunohistochemicalstaining of transduced cells was conducted using a polyclonal goatantibody raised against a peptide, N1 9, mapping at the amino terminusof human GM-CSF (Santa Cruz Biotechnology, Inc.). GM-CSF production wasmeasured in culture medium collected over 3 days in Mx-GM-CSF transducedNIH3T3 and transfected 293T producer cell cultures using commerciallyavailable ELISA kits supplied by R&D Systems, Inc.

Immunoreactive human GM-CSF was noted in 40-50% of transduced NIH3T3cells and 70-80% of transfected 293T cells (n=4 each group), whileGM-CSF production was 32 rig/ml in transduced NIH3T3 cell cultures and100 ng/ml in transfected 293T cell cultures (FIG. 2).

Example 4 In ivo Gene Transfer Studies

In vivo gene transfer studies. were conducted in compliance with aprotocol approved by the University of Southern California InstitutionAnimal Care and Use Committee. To evaluate the efficiency of targetedgene delivery based on the enforced expression of nuclearβ-galactosidase and GM-CSF transgenes in vivo, subcutaneous tumorxenografts were established in 8 week old ˜25 gm athymic nu/nu mice bysubcutaneous implantation of 1×10⁷ MiaPaca2 cells. When the tumors havereached a size of ˜20 mm³, 200 μl of either Mx-nBg marker vector,Mx-GM-CSF vector, a non-targeted CAE-GM-CSF vector, Mx-null or phosphatebuffered saline (PBS, pH 7.4), was injected directly into the tail veindaily for 10 days (cumulative vector dose: 2×10⁷ cfu for each vector).The mice were sacrificed by cervical dislocation one day aftercompletion of one treatment cycle.

Histologic Examination of Harvested Tumor Nodules andImmunohistochemical Analysis for the Presence of β-GalactosidaseTransgene (Gordon et al., 2000b).

Harvested tumor nodules were either quick frozen in liquid nitrogen orfixed in 10% formalin. Formalin-fixed tissue sections were stained withhematoxylin-eosin. Transduction efficiency was determined byimmunohistochemical staining of the tumor nodules, using a mousemonoclonal antibody directed against the β-galactosidase antigen(GAL-40, Sigma, St. Louis Mo., USA) followed by analysis using anOptimas imaging system (Optimas Corporation, Bothell, Wash., USA).Transduction efficiency (expressed as %) is determined by counting thenumber of β-galactosidase positive cells in three high power fields pertumor nodule, divided by the total number of cells×100.

Immunostaining for GM-CSF Protein in Tumor Tissues (Miller et al.,2000).

For detection of the human GM-CSF protein in harvested tumors, tumortissues harvested at the end of the experiment were frozen immediatelyin liquid nitrogen and kept at −70° C. until used. Five μm tissuesections were cut and fixed in ice-cold acetone for 10 min. A goatpolyclonal anti-GM-CSF monoclonal antibody was supplied by Santa CruzBiotechnology, Inc. The slides were blocked with 2% normal goat serumfor 10 min, washed in phophate-buffered saline, and the primary antibodyto GM-CSF diluted at 1:50 was added on the slides for 60 min. Then, theslides were washed three times with PBS and a secondary antibody,anti-goat IgG conjugated with peroxidase (1:100: Vector Laboratories,Burlingame Calif., USA) was added to the slides for 30 min. The slideswere washed five times with PBS and developed with a DAB substrate kit(Vector Laboratories). After counterstain with methyl green, the slideswere examined for presence of brownish-red immunostaining materialindicating presence of the GM-CSF transgene in tumor tissues. Theefficiency of gene delivery (expressed as %) is determined by countingthe number of immunoreactive cells to the anti-GM-CSF antibody in threehigh power fields per tumor nodule, divided by the total number ofcells×100.

Immunoreactive β-galactosidase was noted in ˜35% of cells throughout thetumor nodules of Mx-nBg vector-treated animals (FIG. 3B), while noimmunoreactive protein was noted in the control Mx-null vector-treatedtumors (FIG. 3A). Consistent with the high level transduction of tumornodules noted previously with the Mx-nBg vector, immunreactive humanGM-CSF protein was noted in ˜35% of cells throughout the tumor nodulesof Mx-GM-CSF vector-treated mice (FIG. 4B-C) compared to <1% inCAE-GM-CSF vector-treated and Mx-null vector-treated mice (FIG. 4A).

Example 5 Recruitment of Host Mononuclear Cells into the Tumor Nodulesof Mice Treated with the Mx-GM-CSF Retroviral Vector

Extensive infiltration of host mononuclear cells was noted in tumornodules of Mx-GM-CSF-treated mice (FIGS. 5B&D) compared to minimalmononuclear infiltration in CAE-GM-CSF, Mx-null vector- and PBS-treatedanimals (FIGS. 5A&C). Within the tumor nodule, the tumor infiltratinglymphocyte (TIL) to tumor cell (T) ratio was 1:20 in Mx-GM-CSF-treatedmice compared to 1:90 in nontargeted CAE-GM-CSF vector-treated mice, and1:99 in Mx-null and PBS-treated animals. These findings indicatesuccessful recruitment of host mononuclear cells into the tumor noduleby GM-CSF secreting cells in Mx-GM-CSF vector-treated mice.

Example 6 Bimodal Therapy In Vivo Efficacy Studies

To evaluate bimodal therapy a matrix a targeted retroviral vectorcarrying a cytocidal gene, specifically a dominant negative mutantcyclin G1 gene was constructed using the methods described herein above.The resulting vector is desiganted Mx-dnG1. These studies were conductedin immune competent Balb/c mice (weighing ˜25 gms), in compliance with aprotocol approved by the University of Southern California InstitutionAnimal Care and Use Committee.

To evaluate the efficacy of single versus dual gene therapy in vivo,subcutaneous tumors were established in immune competent Balb/c mice bysubcutaneous implantation of 1×10⁶ murine colon-26 cancer cells (NCI,Bethesda Md., USA). Three days later, 200 ml of either the Mx-dnG1vector (cumulative dose: 1.7×10⁸ cfu/mouse), Mx-GM-CSF vector(cumulative dose: 1.7×10⁸ cfu/mouse), a combination of Mx-dnG1 andMx-GM-CSF vectors (cumulative dose of each vector: 8×10⁷ cfu/fml) or anequivalent volume of phosphate buffered saline (PBS, pH 7.4) placebocontrol was injected intravenously twice a day for 5 days. The size ofthe tumor was measured every 4 days with a Vernier caliper, using theformula for calculating the volume of ellipsoid objects: Tumor Volume,mm³=4/3 p r₁ r₂ r₃. At the end of 5 days, the tumors were resected, andthe animals were given a tumor re-challenge one week after completion ofvector treatment. The significance of differences in mean tumor volumesamong the four treatment groups were evaluated using ANOVA.

To evaluate a dose-dependent response to the Mx-GM-CSF vector in vivo,5×10⁶ cells were implanted subcutaneously, and when the tumors reached asize of ˜35-40 mm³, 200 ml of either a low-dose (cumulative dose: 8×10⁷cfu/mouse) or high-dose Mx-GM-CSF vector (cumulative dose: 1.7×10⁸cfu/mouse), or an equivalent volume of phosphate buffered saline (PBS,pH 7.4) placebo control was injected directly into the tail vein eachday for 16 days. The mice were sacrificed by cervical dislocation oneday after completion of treatment. The significance of differences intumor volumes among the three treatment groups was evaluated usingANOVA.

The combination of the vectors Mx-GM-CSF and Mx-dnG1 induced thegreatest inhibition of tumor growth (p<0.05), and to a lesser extent theMX-dnG1 or Mx-GM-CSF alone. Notably, only half the dose of each vectorwas given to attain the desired anti-tumor effect.

The combination of immunotherapy with radical surgery, chemotherapyand/or radiation therapy has the potential of eradicating minimalresidual disease. In the United States, over 150 approved Phase 1/11gene therapy protocols involve the use of genetically to engineeredsyngeneic or allogeneic cells (tumor cells, T cells, dendritic cells andfibroblasts) for vaccine therapy. Cytokine genes used for immunotherapyinclude IL-1, TNF, IL-2, IFN-y, IL-4, IL-7 and GM-CSF (Foreman et al.,1993; Fearon et al., 1990; Blankenstein et al., 1991; Asher et al.,1991; Tepper et al., 1994; Dranoff et al., 1993; Knobloch et al, 1991;Gansbacher et al., 1991). Other immunostimulatory molecules such as theT-cell co-stimulatory molecule B7.1 (Guinan et al., 1994) or a foreignMHC molecule (Nabel et al., 1993) have also been used to generateanti-tumor immunity.

The major goal of the use of immunostimulatory cytokines is theactivation of tumor-specific T lymphocytes capable of rejecting tumorcells from patients with low tumor burden or to protect patients fromrecurrence of the disease. Treatment of rodents with cancer xenograftswith this strategy have resulted in regression of pre-existing tumorsand cure of the animals from their disease. Further, some animals haveretained immunological memory and resisted a second challenge with theparental tumor cells (Gilboa, 1996; Mackensen et al., 1997).

The mechanisms by which transduced cytokines enhance tumor immunity arethe subject of much investigation. Without being bound by theory, theymay act by increasing the recruitment of cytotoxic T cells able torecognize tumor-specific antigens (e.g. IL-4) (Golumbek et al., 1991) orof CD4-positive cells (e.g. TNF and IL-7) (Asher et al., 1991; Hock etal., 1991), or they may increase tumor antigen expression byupregulating MHC Class I antigens (e.g. IFN-γ) (Watanabe et al., 1989),thereby rendering non-transduced target cells more vulnerable tocytotoxic attack. Alternatively, a combination of these effects may beproduced. For example, IL-2 can recruit both cytotoxic T and NK cellsdirectly and can also induce release of the secondary cytokine IFN-γ(Main et al., 1985), which augments MHC expression on tumor cells (Cozeet al., 1995; Ucar et al., 1995; Handgretinger R et al., 1989).Additional (non-lymphocytic) effector mechanisms appear to contribute aswell, since the tumor site is often infiltrated with eosinophils andmacrophages. As a final mechanism, some cytokines such as GM-CSF mayenhance the activity and differentiation of antigen presenting cells,which ingest tumor cells and present their antigens either inassociation with Class I or Class II MHC molecules, thereby recruitingCD8+ or CD4+ tumor specific T lymphocytes (Dranoff et al., 1993).

The inhibition of subcutaneous tumor growth in nude mice by intravenousadministration of a matrix-targeted retroviral vector bearing acytocidal/cytostatic cyclin G1 construct (Gordon et al., 2000a) wasrecently reported. These retroviral vectors displayed a collagen-bindingmotif derived from von Willebrand coagulation factor that could targetextracellular matrix (ECM) exposed during tumor growth, angiogenesis,and metastasis. Enhanced vector penetration and transduction of tumornodules was demonstrated and correlated with therapeutic efficacywithout associated toxicity. In the present study, we demonstrated highlevel GM-CSF secretion in NIH3T3 and 293T cell cultures transduced ortransfected, respectively, with a matrix-targeted retroviral vectorbearing a human GM-CSF construct, as well as high level transduction ofsubcutaneous human cancer xenografts in nude mice by repeatedintravenous injections of the Mx-GM-CSF vector. Further, the enforcedexpression and secretion of GM-CSF by transduced cells throughout thetumor nodule resulted in recruitment of host mononuclear cells into thetumor nodule. These studies were conducted in athymic nude mice lackingcytotoxic T cells. In immune competent mice with a complete repertoireof T lymphocytes and antigen-presenting B cells, dendritic cells andmacrophages, regression of the tumor nodules and establishment of longterm antitumor immunity would be expected to occur, particularly whentumor antigens are presented simultaneously by concomitantadministration of a matrix-targeted vector bearing a cytocidal gene.

The data demonstrates that a targeted retroviral vector comprising acytokine gene, such as a matrix (i.e. collagen)-targeted retroviralvector bearing a human GM-CSF construct can be injected intravenously toachieve (i) high level efficiency of cytokine gene delivery into solidtumors, (ii) secretion of GM-CSF by tumor cells within the solid tumor,and (iii) recruitment of host mononuclear cells (tumor infiltratinglymphocytes, TIL) into the GMCSF secreting tumor nodules for eradicationof primary, metastatic, inaccessible or minimal residual disease.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand the following examples are intended to illustrate and not limit thescope of the invention. Other aspects, advantages and modificationswithin the scope of the invention will be apparent to those skilled inthe art to which the invention pertains.

REFERENCES

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1.-10. (canceled)
 11. A pharmaceutical composition comprising a targetedretroviral vector particle comprising a modified viral surface proteinfor targeting the vector and a cytokine, and a second targetedretroviral vector particle comprising a modified viral surface proteinfor targeting the second vector and a cytocidal gene.
 12. Thepharmaceutical composition of claim 11, wherein the second targetedretroviral particle is targeting the extracellular matrix or tumorvasculature.
 13. The pharmaceutical composition of claim 11, wherein thecytocidal gene is selected from the group consisting of tumor suppressorgenes, thymidine kinases or mutated cyclin genes.
 14. The pharmaceuticalcomposition of claim 13, wherein the mutated cyclin gene is a dominantnegative mutation of a cyclin G1 gene.
 15. A method for inhibitingcancer in a subject comprising administering to the subject an effectiveamount of the pharmaceutical composition of claim
 11. 16. A compositioncomprising a targeted retroviral vector particle comprising a modifiedviral surface protein for targeting the vector to the Von Willebrandcoagulation factor and a cytokine gene and a targeted retroviral vectorparticle comprising a modified viral surface protein for targeting thevector to the Von Willebrand coagulation factor and a cytocidal gene.17. The composition of claim 16, wherein the cytocidal gene is a mutatedcyclin gene.
 18. The composition of claim 17, wherein the cytocidal geneis a dominant negative mutation of the cyclin G1 gene.
 19. Thecomposition of claim 16, wherein the cytokine is selected from the groupconsisting of IL-1, TNF, IL-2, IFN-γ, IL-4, IL-7 and GM-CSF.
 20. Thecomposition of claim 19, wherein the cytokine is GM-CSF.
 21. Thecomposition of claim 16, further comprising a pharmaceutical excipient.22. A method for inhibiting cancer in a subject comprising administeringto the subject an effective amount of the pharmaceutical composition ofclaim 21.